| Avian Influenza (AI) outbreak constantly around the world, which brought greateconomic losses to the poultry industry since it has been found. The hazards of lowpathogenic avian influenza H9subtype have aroused great attention in recent years. Inorder to understand the immunogenicity, cell culture and molecular biology characteristicsof Henan H9subtype, the following research has been undertaken.The2strains viruses were isolated by the SPF chicken embryo from chicken visceraltissues suspected of being infected with AIV H9subtype in Luohe and Zhengzhou city in2011. They were detected by HA, HI and RT-PCR method.Another2strains AIV H9subtype viruses were isolated and identification in2001. The test was carried out to awareof its cross immunogenicity protection. Inactivated vaccines were prepared with2strains ofAIV H9subtype isolated in2001, and used to inoculate SPF chickens. The4strains wereused to infect immunized chickens. The results indicated that the HI antibody titers againstH9subtype were significantly increased after inoculation, and there was a good crossprotection force between H9subtype isolated from different periods and locations. Thestudy demonstrated that the inactivated vaccine prepared with the isolated strains in2001offered good protection against current pandemic H9subtype.To inoculate MDCK cells with H9subtype avian influenza, the condition of trypsinconcentration, serum concentration, adsorption time, inoculation amount, the passagenumber and time for virus harvest were optimized. The corresponding haemagglutinintiters (HA) were detected. The results showed that: the optimal trypsin concentration,serum concentration, adsorption time, inoculation amount, and time for virus harvest forculture of influenza virus were1.0μg/mL,0,90min,10-1dilution of viruses produced inchick embryo and3d respectively. The virus hemagglutinin titers cultured under theoptimized condition could reach7. After subculture in MDCK cells for4passages,the HAtiter of the two strains isolated from LuoHe decreased to0.However, the two strainsisolated from Zhengzhou could be passaged in MDCK cells more than ten times in a row.ZZ11strains could continue passage in MDCK cells for more than45times.Make a sequence analysis of HA and NA genes of the4isolated AIV H9subtype. Andthe results showed that, HA gene of the LH11strain was1707bp, the maximum ORF was1518bp, which was34-1716nucleotides encoding505amino acids.While for the other three, HA genes were1742bp, full length of ORFs were1683bp, which were34-1716nucleotides encoding560amino acids. Homologies of the HA genes nucleotides and aminoacids were92.5%~98.8%, and93.3%~97.9%respectively. In addition to LH01strain,NAgenes were1469bp. NA gene of the LH01strain was1470bp.All the ORFs of NA geneswere1401bp, which were20-1420nucleotides encoding466amino acids. Homologies ofthe NA genes nucleotides and amino acids were93.4%~99.7%and93.6%~99.4%respectively. Make an analysis of the amino acids of the AIVs about the pathogenicity andhost-selecitivity. The amino acids of the HA cleavage site were PSRSSR↓GLF andPVRSSR↓GLF respectively, in line with the characteristics of the low pathogenic avianinfluenza virus. HA genes receptor binding site of4isolates except234, the remainingseven bits were consistent, of which109bits,161bits,163bits,191bits,202bits,203bitswere consistent with four representative strain.The198amino acids of4isolates wasconsistent with CK/BJ/1/94representative strain, while the CK/HK/G9/97andCK/HK/G23/97were A, and the DK/HK/Y280/97was T. The226amino acids of allisolates were G, which was the description that the hosts of all isolates have not change.However, the234amino acids of the Zhengzhou isolates were L showing the bindingproperties of human influenza receptors. LH11strain had five glycosylation sites, andLH01strain had seven glycosylation sites. Zhengzhou isolates had eight glycosylationsites.The315amino acids of Luohe isolates were P, while Zhengzhou isolates were S.Thismutation result that the Zhengzhou isolates increase a glycosylation site. There were I, T, Edeletion on62,63and64amino acids of NA stem Cap of4isolates, resulted in the loss ofthe61glycosylation sites. The highly conservation of86,146,200,234sites of amino acidsof NA protein was essential in the neuraminidase active. The45amino acids of Luoheisolates were L, while representative strains were p, resulted in increasing a glycosylationsite. The Zhengzhou isolates due to the264amino acids mutation as N, while therepresentative strains were H, so here add a potential glycosylation site. The ZZ11strainsdue to the368amino acid mutation as N, while the representative strains were K or E, sohere add a potential glycosylation site.The ZZ01strain due to the402amino acid mutationas S,while representative strains were N, resulted in the loss of a glycosylation site.HA and NA genes sequence analysis of the ZZ11strains produced in chick embryoand the cells. Homologies of the HA genes nucleotides and amino acids were99.3%～99.6%and98.8%～99.3%respectively. Homologies of the NA genes nucleotides andamino acids were99.7%～99.9%and99.1%～99.8%respectively. Nucleotidehomologies were above99.3%, and amino acid homologies were above98.8%, whichexplained passaging in MDCK cells was very stable. The HA and NA genes phylogenetic tree analysis showed the closest evolutionaryrelationship with HA gene was the DK/HK/Y280/97, which belonged to Y280formationsin Eurasian lineage, and the closest evolutionary relationship with NA gene was aslo theDK/HK/Y280/97.These results provided the technical basis for avian influenza virus prevention andseparation method of Henan AIV H9subtype... |
PDF Full Text Request